Long non-coding RNA growth arrest specific transcript 5 acting as a sponge of MicroRNA-188-5p to regulate SMAD family member 2 expression promotes myocardial ischemia-reperfusion injury

Abstract

The purpose of this work is to probe into the potential role of long non-coding RNA growth arrest specific transcript 5 (lncGAS5)/ microRNA (miR)-188-5p/SMAD2 axis in MIRI. Through ligating the left anterior descending (LAD) coronary artery, MIRI animal model and hypoxia/reoxygenation (H/R) myocardial injury model in vitro were established. Via adenovirus or plasmid transfection, lncGAS5/MiR-188-5p/SMAD2 expression was up-regulated or down-regulated in the study. RT-qPCR was applied to check LncGAS5/MiR-188-5p/SMAD2 mRNA expression, HE staining for histopathological staining, TUNEL staining and flow cytometry to examine cardiomyocyte apoptotic rate, CCK-8 to check cell viability, ELISA to detect inflammatory factor levels, Western blot to examine Bax, Bcl-2, cleaved caspase-3, NF-κB and SMAD2 expression, and dual luciferase reporter experiment to examine the targeting relationship of miR-188-5p with LncGAS5 and SMAD2. The results indicated that LncGAS5 and SMAD2 were highly expressed in MIRI and miR-188-5p was under-expressed. Silencing LncGAS5 and SMAD2 or overexpressing miR-188-5p could reduce MIRI in myocardial tissue, cardiomyocyte apoptosis, inhibit Bax, cleaved caspase-3 and NF-κB expressions and promote Bcl-2 expression, while reducing inflammatory factors TNF -α, IL-1β and IL-6 levels. Overexpressing LncGAS5 promoted MIRI. Additionally, the impact of silencing LncGAS5 on MIRI could be reversed through inhibiting miR-188-5p. LncGAS5 acted as a sponge of miR-188-5p to target SMAD2 expression. In conclusion, Silencing LncGAS5 is available to improve MIRI through regulating miR-188-5p/SMAD2 axis, and may be used as a potential target for treating MIRI in the future.

Keywords: Long non-coding RNA growth arrest specific transcript 5; NF-ΚB; microRNA-188-p; myocardial ischemia-reperfusion injury; smad2.

Figure 1.

Expression of lncGAS5 in clinical samples.LncGAS5 expression in the blood of AMI patients before and after PCI was detected by RT-qPCR; one-way ANOVA was applied to calculate the significance of each group; the variance was corrected via Tukey’s test. *P < 0.05

Figure 2.

LncGAS5 is upregulated in MIRI and silencing LncGAS5 inhibits MIRI.A. RT-qPCR to detect LncGAS5 expression in mice myocardial tissues in the Sham, the MIRI, the sh-NC, and the sh-LncGAS5 groups; B. HE staining representative images of in mice myocardial tissues in the Sham, the MIRI, the sh-NC, and the sh-LncGAS5 groups; C. TUNEL staining to in mice myocardial tissue apoptosis in the Sham, the MIRI, the sh-NC, and the sh-LncGAS5 groups; D. Western blot to check Bax, Bcl-2 and lysed caspase-3 expressions in mice myocardial tissues in the Sham, the MIRI, the sh-NC, and the sh-LncGAS5 groups; E. ELISA to examine TNF-α, IL-1β and IL-6 levels in mice myocardial tissues in the Sham, the MIRI, the sh-NC, and the sh-LncGAS5 groups; the value was expressed as mean ± SD (n = 3); one-way ANOVA was applied to calculate the significance of each group; the variance was corrected via Tukey’s test. *P < 0.05

Figure 3.

Overexpressing LncGAS5 promotes MIRI.A. RT-qPCR to detect LncGAS5 expression in HL-1 cells in the Control, the H/R, the oe-NC, the oe-LncGAS5 groups; B. CCK-8 to check HL-1 cell viability in the Control, the H/R, the oe-NC, the oe-LncGAS5 groups; C. Flow cytometry to check HL-1 cell apoptosis in the Control, the H/R, the oe-NC, the oe-LncGAS5 groups; D. Western blot to check Bax, Bcl-2 and lysed caspase-3 expressions in HL-1 cells in the Control, the H/R, the oe-NC, the oe-LncGAS5 groups; E. ELISA to examine TNF-α, IL-1β, and IL-6 levels in HL-1 cells in the Control, the H/R, the oe-NC, the oe-LncGAS5 groups; F. Western blot to examine NF-κB p65 expression in HL-1 cells in the Control, the H/R, the oe-NC, the oe-LncGAS5 groups; the value was expressed as mean ± SD (n = 3); one-way ANOVA was applied to calculate the significance of each group; the variance was corrected via Tukey’s test. *P < 0.05

Figure 4.

Upregulating miR-188-5p inhibits MIRI.A. RT-qPCR to detect LncGAS5 expression in HL-1 cells in the Control and the H/R groups; B. RT-qPCR to detect LncGAS5 expression in HL-1 cells in the mimic NC and the miR-188-5p mimic groups; C. CCK-8 to check HL-1 cell viability in the mimic NC and the miR-188-5p mimic groups; the si-NC and the si-SMAD2 groups; D. Flow cytometry to check HL-1 cell apoptosis in the mimic NC and the miR-188-5p mimic groups; E. Western blot to check Bax, Bcl-2 and lysed caspase-3 expressions in HL-1 cells in the mimic NC and the miR-188-5p mimic groups; F. ELISA to examine TNF-α, IL-1β, and IL-6 levels in HL-1 cells in the mimic NC and the miR-188-5p mimic groups; G. Western blot to examine NF-κB p65 expression in HL-1 cells in the mimic NC and the miR-188-5p mimic groups; the value was expressed as mean ± SD (n = 3); one-way ANOVA was applied to calculate the significance of each group; the variance was corrected via Tukey’s test. *P < 0.05

Figure 5.

LncGAS5 acts as a sponge of miR-188-5p to regulate SMAD2 expression.A. RT-qPCR to detect miR-188-5p expression in mice cardiomyocytes in the sh-NC and the sh-LncGAS5 groups; B. RT-qPCR to detect miR-188-5p expression in HL-1 cells in the oe-NC and the oe-LncGAS5 groups; C. Bioinformatics website http.//starbase.sysu.edu.cn/ to predict the potential-binding sites of LncGAS5 and miR-188-5p; D. Dual luciferase reporter experiment to check targeting relationship between LncGAS5 and miR-188-5p; E. RT-qPCR and western blot to detect SMAD2 expression in mice myocardial tissue in the Sham and the MIRI groups as well as in HL-1 cells in the Control and the H/R groups; F. RT-qPCR and western blot to detect SMAD2 expression in HL-1 cells in the mimic NC and the miR-188-5p mimic groups; G. Bioinformatics website http://starbase.sysu.edu.cn/ to predict potential-binding sites of SMAD2 and miR-188-5p; H. Dual luciferase reporter experiment to check targeting relationship between SMAD2 and miR-188-5p; the value was expressed as mean ± SD (n = 3); one-way ANOVA was applied to calculate the significance of each group; the variance was corrected via Tukey’s test. *P < 0.05

Figure 6.

Silencing SMAD2 inhibits MIRI.A. RT-qPCR and western blot to detect SMAD2 expression in HL-1 cells in the si-NC and the si-SMAD2 groups; B. CCK-8 to check HL-1 cell viability in the si-NC and the si-SMAD2 groups; C. Flow cytometry to check HL-1 cell apoptosis in the si-NC and the si-SMAD2 groups; D. Western blot to check Bax, Bcl-2 and lysed caspase-3 expression in HL-1 cells in the si-NC and the si-SMAD2 groups; E. ELISA to examine TNF-α, IL-1β and IL-6 levels in HL-1 cells in the si-NC and the si-SMAD2 groups; F. Western blot to examine NF-κB p65 expression in HL-1 cells in the si-NC and the si-SMAD2 groups; the value was expressed as mean ± SD (n = 3); one-way ANOVA was applied to calculate the significance of each group; the variance was corrected via Tukey’s test. *P < 0.05

Figure 7.

Up-regulated SMAD2 facilitates MIRIA. SMAD2 expression in HL-1 cells of the oe-NC and the oe-SMAD2 groups detected by RT-qPCR and western blot; B. HL-1 cell viability in the oe-NC and the oe-SMAD2 groups examined by CCK-8; C. The apoptosis of HL-1 cells in the oe-NC and the oe-SMAD2 groups detected via flow cytometry; D. The expression of Bax, Bcl-2 and cleaved caspase-3 in HL-1 cells of the oe-NC and the oe-SMAD2 groups examined by western blot; E. Levels of TNF-α, IL-1β and IL-6 in HL-1 cells of the oe-NC and the oe-SMAD2 groups detected by ELISA; F. The expression of NF-κB p65 in HL-1 cells of the oe-NC and the oe-SMAD2 groups examined by western blot. The value was expressed as mean ± SD (n = 3); one-way ANOVA was applied to calculate the significance of each group; the variance was corrected via Tukey’s test. *P < 0.05

Figure 8.

LncGAS5 regulates MIRI development through miR-188-5p/SMAD2 axis.A. RT-qPCR to detect SMAD2 expression in HL-1 cells in the oe-NC and the oe-LncGAS5 groups; B. RT-qPCR to detect SMDA2 expression in HL-1 cells in the H/R, the si-LncGAS5 + inhibitor NC, and the si-LncGAS5 + miR −188-5p inhibitor groups; C. CCK-8 to check HL-1 cell viability in the H/R, the si-LncGAS5 + inhibitor NC, si-LncGAS5 + miR-and 188–5p inhibitor groups; D. Flow cytometry to check HL-1 cell apoptosis in the H/R, the si-LncGAS5 + inhibitor NC, and the si-LncGAS5 + miR-188-5p inhibitor groups; E. Western blot to check Bax, Bcl-2 and lysed caspase-3 expressions in HL-1 cells in the H/R, the si- LncGAS5 + inhibitor NC, and the si-LncGAS5 + miR-188-5p inhibitor groups; F. ELISA to examine TNF-α, IL-1β and IL-6 levels in HL-1 cells in the H/R, the si-LncGAS5 + inhibitor NC, and the Si-LncGAS5 + miR-188-5p inhibitor groups; G. Western blot to examine NF-κB p65 expression in HL-1 cells in the H/R, the si-LncGAS5 + inhibitor NC, and the si-LncGAS5 + miR-188-5p inhibitor groups; the value was expressed as mean ± SD (n = 3); one-way ANOVA was applied to calculate the significance of each group; the variance was corrected via Tukey’s test. *P < 0.05